Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery.

HHS Public Access Author manuscript Author Manuscript

Curr Top Med Chem. Author manuscript; available in PMC 2017 February 22. Published in final edited form as: Curr Top Med Chem. 2017 ; 17(1): 4–15.

Targeting Non-Catalytic Cysteine Residues Through StructureGuided Drug Discovery Kenneth K. Hallenbecka, David M. Turnera, Adam R. Rensloa, and Michelle R. Arkina,* aSmall

Molecule Discovery Center and Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA

Author Manuscript

Abstract

Author Manuscript

The targeting of non-catalytic cysteine residues with small molecules is drawing increased attention from drug discovery scientists and chemical biologists. From a biological perspective, genomic and proteomic studies have revealed the presence of cysteine mutations in several oncogenic proteins, suggesting both a functional role for these residues and also a strategy for targeting them in an ‘allele specific’ manner. For the medicinal chemist, the structure-guided design of cysteine-reactive molecules is an appealing strategy to realize improved selectivity and pharmacodynamic properties in drug leads. Finally, for chemical biologists, the modification of cysteine residues provides a unique means to probe protein structure and allosteric regulation. Here, we review three applications of cysteine-modifying small molecules: 1) the optimization of existing drug leads, 2) the discovery of new lead compounds, and 3) the use of cysteine-reactive molecules as probes of protein dynamics. In each case, structure-guided design plays a key role in determining which cysteine residue(s) to target and in designing compounds with the proper geometry to enable both covalent interaction with the targeted cysteine and productive noncovalent interactions with nearby protein residues.

Keywords Non-catalytic cysteine; Covalent drugs; Structure-based design; Chemical probes; disulfide Tethering; Lead optimization; Protein dynamics; Protein allostery

1. INTRODUCTION Author Manuscript

Cysteine is an underrepresented residue in protein sequence (3.3% frequency [1]) but is disproportionately involved in protein function, with >50% of cysteine residues being solvent exposed and implicated in a myriad of biochemical processes [2]. Cysteine serves as the reactive nucleophile in many hydrolases (such as cysteine proteases) and can mediate redox reactions (e.g., protein disulfide isomerase). Oxidized forms of cysteine with sulfenic acid or nitrosothiol functionality are increasingly appreciated as playing a role in cellular signaling, and this suggests the possibility of targeting such oxidized forms with specific *

Address correspondence to this author at the Department of Pharmaceutical Chemistry, University of California, San Francisco, Box 2552, San Francisco, CA 94158, USA; Tel/Fax: ++1-415-514-4313, +1-415-514-4504; [email protected]. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflict of interest.

Hallenbeck et al.

Page 2

Author Manuscript

small molecules [3–4]. Finally, disulfide bond formation between two cysteine residues has been long recognized as contributing to protein tertiary structure. Taken together, these features make cysteine an attractive target for modification by small molecules. Here, we focus on approaches to target non-catalytic cysteine residues.

Author Manuscript

The concept behind covalent modification of cysteine residues is schematized in Fig. (1). An initial non-covalent complex (E*I) positions the electrophilic group within range of the nucleophilic thiol moiety and facilitates bond formation (k2). For truly irreversible inhibitors, the resulting covalent complex (E-I) remains intact; however, for reversible electrophiles (e.g. disulfides), the ligand-bound complex dissociates (k−2) over time to reform the initial non-covalent complex (E*I). Thus, cysteine-modifying drugs rely on two binding interactions – covalent and non-covalent – that can be independently and iteratively optimized to obtain the necessary selectivity and potency to be useful chemical probes or drug leads. Cysteine-modifying compounds have been directed at both catalytic and non-catalytic residues. Modifying catalytic cysteines, such as those found in deubiquitinases and caspases, have an obvious impact on enzyme function. However, catalytic residues in enzyme active sites are generally highly conserved within families, and isoform selectivity can be difficult to achieve. Non-catalytic cysteines are generally less conserved, making them attractive for selective target modulation. Chemical proteomic studies employing activity-based probes have identified various reactive, functional, and non-catalytic cysteine residues whose functions could be probed and modulated with drug-like covalent molecules. These studies have revealed that inherent thiol reactivity spans six orders of magnitude [5], an observation that is gergermane in any effort to develop highly selective cysteine-targeted compounds.

Author Manuscript

As appreciation for the targetable nature of cysteine residues has grown, covalent approaches to drug discovery are also resurgent. However, covalent pharmacology inevitably raises the concern that reactive drugs or drug metabolites can induce organ damage or evoke an immune response through off-target (nonselective) binding to other proteins [6–7]. A related concern is that an electrophilic drug can lose activity because it is rapidly inactivated and eliminated via reaction with native nucleophiles (e.g. GSH) [6, 8].

Author Manuscript

These arguments are often countered by noting that many safe and well tolerated drugs in use for decades, such as aspirin and penicillin, act via covalent modification of their targets [9–10] and that the intentional targeting of nucleophilic sites with appropriately tuned electrophiles can mitigate the risk of covalent pharmacology. Giving appropriate attention to these potential issues is likely a factor in the recent clinical successes of covalent drug candidates [11]. Taking the advantages and challenges into account, the design of selective cysteinemodifying molecules as drug leads or as chemical probes for biomolecules has proven an attractive approach for the following distinct applications: 1.

Lead optimization. Covalent pharmacology can enhance potency and selectivity of lead compounds, most compellingly by increasing target residence time. Covalent pharmacology is typically associated with ‘durable’ target inhibition in

Curr Top Med Chem. Author manuscript; available in PMC 2017 February 22.

Hallenbeck et al.

Page 3

Author Manuscript

vivo, and often very different pharmacokinetic/ pharmacodynamic (PK/PD) relationships as compared to drugs exhibiting reversible, fast-off inhibition kinetics [6]. Selectivity for protein isoforms containing the targeted residue is another benefit of this approach for lead optimization. Nearly any cysteine proximal to a known drug-binding site is a potential candidate. Knowledge of the structure, to support computational-guided design of the cysteine-reactive analog, is also highly desirable.

Author Manuscript

2.

Chemical handles to identify new lead scaffolds. In targets where structural data indicates a binding pocket is available near a solvent-exposed cysteine, screening libraries of diverse molecules containing a cysteine-reactive moiety is an effective strategy for lead discovery. Computational approaches help triage promising target sites and identify scaffolds around which to build electrophile libraries for screening. One particularly interesting application for new drug discovery is targeting cysteine mutations found in oncogenic proteins; cysteine reactive molecules could first validate the function of these mutations in disease, then serve as lead compounds for therapeutic development.

3.

Site-specific study of protein allostery, dynamics, and structure-function relationships. Native or engineered surface cysteines can be used to find molecules that bind at known sites of allosteric regulation, or can uncover previously undetected (‘cryptic’) binding pockets.

2. IMPROVING DRUG PROPERTIES FOR KNOWN SCAFFOLDS 2.1. Kinases

Author Manuscript Author Manuscript

It is startling to recall that twenty-five years ago, kinases were considered ‘undruggable’ targets. The central importance of kinases and the high structural homology within the family suggested that imperfect selectivity would lead to unacceptable levels of toxicity. Through the creative efforts of many laboratories, kinase inhibitors are now a wellestablished class of cancer therapeutics. However, the development of drug resistance during kinase-inhibitor therapy is also common. First-generation inhibitors of epidermal growth factor receptor (EGFR) were effective in treating certain subtypes of lung carcinoma, but a mutation in the gatekeeper residue (T790M) resulted in a steric clash in the binding site (Fig. 2A) ultimately leading to clinical relapse [12–13]. Walter and colleagues demonstrated that EGFR Cys797, which sits at the edge of the ATP-binding pocket and is present in just 2% of kinases, could be targeted to improve potency and recover function in the presence of T790M [14]. Selectively targeting a rare cysteine to increase drug residence time was expected to result in an improved clinical outcome (Fig. 2A, B). However, the effectiveness of second-generation EGFR inhibitors was limited by on-target toxicity, since the drugs inhibited both mutant and WT EGFR [15–16]. To selectively target T790M EGFR, the 1st and 2nd generation quinazoline scaffold was replaced by other heterocyclic scaffolds into which an acrylamide electrophile could be readily introduced [17]. Several pyrimidine-based molecules were identified that exhibited 30 to 100-fold selectivity for T790M over WT EGFR. One of these pyrimidines, Osimertinib [18] was approved by the FDA in November 2015, while and Rociletinib [19], was not approved for treatment of non-small-cell lung


Hallenbeck et al.

Page 4

Author Manuscript

cancer [20–21]. The search for the next generation of covalent EGFR inhibitors continues, guided by rational design and lessons learned from the clinic [22]. As these examples illustrate, the Michael reaction between cysteine thiol as nucleophile and an alpha-beta unsaturated carbonyl (e.g. acrylamide) has figured prominently in the design of covalent kinase inhibitors. Michael ‘acceptors’ are attractive for these applications because their reactivity can be tuned by changing the nature of the electron-withdrawing carbonyl (or related) function and/or by altering the steric environment surrounding the electrophilic beta carbon atom.

Author Manuscript

The early success of covalent EGFR inhibitors motivated use of the approach in many other kinases. Ibrutinib, which received a breakthrough drug designation in 2013 for mantle cell lymphoma, del17p chronic lymphocytic leukemia and Waldenstrom's macroglobulinemia [23], contains an acrylamide warhead that irreversibly modifies Cys481 in Bruton’s tyrosine kinase (BTK). Ibrutinib’s scaffold was identified in a screen and was prioritized because it showed selectivity for a small group of Tec and Src-family kinases. Sequence comparison and structural homology modeling suggested that BTK contained a nucleophilic cysteine in the position analogous to Cys797 in EGFR [24]. This observation motivated a structureguided medicinal chemistry effort that sampled three potential Michael acceptors – propiolamide, vinyl sulfonamide, and acrylamide – and found the last had the best activity (0.5 nM against BTK) and selectivity profile.

Author Manuscript

The Janus kinase (JAK) family have >80% amino acid identity in their ATP-binding site, exemplifying the kinase selectivity problem. Because of their importance to cytokine signaling pathways and potential as autoimmune disease therapeutics, many pan-JAK inhibitors have been described [25]. However, no reversible, isoform-specific inhibitors exist. JAK3 is the only JAK that contains a cysteine (Cys909) at the EGFR and BTK site, and was therefore targeted with tricyclic JAK inhibitors that included a terminal electrophile designed to irreversibly react with JAK3 Cys909 [26]. These compounds inhibited JAK3 with 104-fold) over common serine proteases that bind boronic acids. 3.2. Experimental Library Design

Author Manuscript

Assembly of cysteine-reactive small molecule libraries for experimental screening has tended to use a fragment-based philosophy [41–42]. Fragment-based drug discovery (FBDD) seeks to identify low molecular weight fragments (typically 90% at 1 µM, and each of these sensitive kinases possessed the analogous C481. Importantly, other C481 kinases, including EGFR and JAK3 that were regarded as possible off-targets, were relatively unaffected (IC50 > 3 µM). Finally, this compound was evaluated


Hallenbeck et al.

Page 11

Author Manuscript

for BTK inhibition in vivo and was found to retain target engagement >24 hours after oral dosing, by which time free inhibitor had been cleared from circulation. Overall, the tunability of slowly reversible warheads and the corresponding benefits in terms of selectivity and in vivo PD properties make a convincing case for the wider application of this approach in drug discovery [79].

4. EXPLORING ALLOSTERY

Author Manuscript

Cysteine-targeted agents have also served as chemical-biology tools to explore protein conformation and allostery. Conformational changes, e.g., caused by posttranslational modification, protein-protein interactions, and the binding of signaling molecules, play a critical role in protein function. Designing molecules that affect a specific protein state could make for highly selective drugs. However, compounds that act at allosteric sites on proteins are often found serendipitously. Allosteric sites can also be cryptic sites, in that they are not apparent in crystal structures of the unbound protein and are therefore difficult to discover and model computationally. On the other hand, some cryptic sites might not bind endogenous ligands, yet might have nascent allosteric potential that synthetic ligands could harness. For these reasons, predicting and evaluating the functional relevance of cryptic sites are active areas of research, and the potential to proactively identify and target allosteric and/or cryptic sites remains an important challenge for structure-based drug discovery. 4.1. Native Cysteine Residues


In the case of caspases, disulfide trapping identified a previously unknown allosteric site. Caspase-7 is a dimeric cysteine protease and a potent effector of cell death by apoptosis. The enzyme is expressed as an inactive dimeric zymogen that is cleaved under apoptotic conditions to the active form; a series of loop movements causes a significant change in the overall conformation of the active sites and the dimer interface. Disulfide tethering identified two compounds called FICA and DICA (Fig. 3D,E) that bound selectively to the Cys290 residue at the dimer interface and stabilized a zymogen-like, inactive conformation of the ‘active’ caspase-7 [80]. Caspase-1, a caspase involved in proinflammatory processes, was also found to have a cysteine residue (Cys331) at the dimer interface that could be targeted by a disulfide-containing compound (Fig. 3F,G) [81]. Each of these disulfide-trapped compounds bound with two molecules tethered to symmetry-related cysteines at the interface, but the compounds bound with different orientations; for instance, compound 34 made compound-compound interactions in the caspase-1 site (Fig. 3G), while the two bound DICA molecules did not interact (Fig. 3D). For both proteins, analysis of the structures of the compound-trapped inactive state and the active conformation uncovered an allosteric network over the 15A between the allosteric site and the enzyme active site [82–83]. In an interesting and potentially generalizable application, compound 34-bound caspase-1 was used to select anti-caspase-1 antibodies that preferentially bound to the inactive conformation [84]. 4.2. Engineered Cysteine Residues Naturally, many allosteric or cryptic sites will not have native cysteine residues nearby. In these cases, engineering cysteine residues and probing them with cysteine-reactive


Hallenbeck et al.

Page 12

Author Manuscript

compounds can provide deep insight into protein structure and dynamics. The surface of the protein hormone IL-2 was probed with a series of cysteine residues followed by disulfide trapping. IL-2 is a four-helix bundle that binds to a trimeric receptor. Eleven cysteine mutations were made, one-at-a-time, along the surface of the alpha-chain binding site of IL-2 [38]. This face of IL-2 was found to be amphiphilic, with one side being hydrophilic, flat, and structurally stable, while the other side was more hydrophobic and structurally dynamic. Disulfide screening identified many more compounds that bound to the dynamic portion of the interface, in some cases trapping conformations not seen in structures of the apo protein, including conformations induced by the binding of ligands at other locations on the surface of IL-2. Thus, protein-protein interfaces can have regions of structural adaptivity, which might have a role in binding multiple protein partners [85] and/or may be exploited by small-molecule ligands [38, 86].


Protein-protein interactions can also allosterically regulate enzyme activity, as is the case with different classes of kinases. The AGC kinases, for instance, have a common allosteric site in the N-lobe of the kinase domain, where substrate proteins or regulatory domains of the kinase itself can bind [87]. Binding to this allosteric site co-localizes the substrate and enzyme and also allosterically activates catalysis by positioning the regulatory ‘C helix’ into an active position. The AGC kinase PDK1 is a well-studied example of this allosteric regulation, and multiple small-molecule modulators that bind to the allosteric site have been designed. Cysteine mutagenesis followed by disulfide trapping identified several compounds that allosterically activated or inhibited kinase activity [88]. Importantly, both inhibitors and activators could be selected at the same cysteine residue; hence, the details of the noncovalent binding interactions and molecular shape determined allosteric outcome, not structural changes in the protein due to cysteine mutagenesis per se. X-ray structures of an activator and inhibitor bound to PDK1 (Fig. 3H) highlighted the mechanism of allostery; the smaller compound (Fig. 3J) stabilized a conformation in which the regulatory C-helix was pulled away from the active site, while the larger compound (Fig. 3I) pushed the Chelix down and into the active conformation [88]. These studies underscore the subtlety between binding and allostery that makes small-molecule design of allosteric modulators both fascinating and complicated.

Author Manuscript

Bishop and coworkers have taken a protein engineering approach to develop selective allosteric inhibitors of protein tyrosine phosphatase (PTP) enzymes [89]. This important class of signaling enzymes has encountered significant challenges for drug discovery, given the similarity of the active site across the family and the challenge with obtaining cellpermeable active-site inhibitors. The Bishop lab found that the PTP Shp2 was sensitive to inhibition by the cysteine-reactive dye FLAsH, which binds to 2-, 3-, or 4 cysteine residues. They identified two nearby cysteine residues, Cys333 and Cys367 that were buried in the apo-structure of Shp2, but became surface accessible in the presence of FLAsH [89]. Cys333 is unique to Shp2 among PTPs, potentially providing a novel therapeutic strategy for targeting Shp2. However, FLAsH itself did not bind tightly enough to wild type Shp2 to bind selectively in cell lysates, so the investigators engineered an additional Cys368 to provide trivalent coordination of FLAsH [90]. Intriguingly, they were able to create similar allosteric sites by engineering cysteine residues at the analogous position in several PTPs. Thus, they


Hallenbeck et al.

Page 13

Author Manuscript

discovered a novel, cryptic allosteric site that can be used to probe the functions of specific PTPs in cells.

Author Manuscript

Cysteine mutagenesis/reactivity has also been used to experimentally validate potential hidden/cryptic allosteric sites identified computationally. Bowman, et al. utilized a Markov state model that evaluated protein structural changes on the microsecond to millisecond timescale [91]. Using a drug-resistant mutant of TEM-1 beta-lactamase as a model system, they collected an ensemble of protein structures and looked for transient pockets that a) were fragment-to-lead sized, b) correlated with motions at the active site, and c) included residues that changed from buried to surface-exposed upon pocket formation. They then mutated these residues to cysteine and used the thiol-detection reagent 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) to demonstrate that they could be labeled. The rate of DTNB reaction with cysteine mutants supported the hypothesis that pockets opened and closed transiently. In three cases, TNB-labeled enzyme had a reduced catalytic efficiency, suggesting that trapping these pockets did have an allosteric effect on the active site. The authors envisaged a pipeline in which cryptic pockets would be identified computationally, then validated through cysteine mutation and screening with cysteine-reactive libraries. Structure-guided design could then be used to optimize the compounds so that they allosterically inhibit the noncysteine containing protein.

CONCLUSION

Author Manuscript

The post-translational modification (PTM) of reactive amino acid side chains is well recognized as an essential mechanism by which biology regulates cell signaling, protein structure, and the epigenetic control of gene expression. The various examples and approaches to cysteine modification described herein might be considered as examples of unnatural PTM leveraging the vastly greater access to chemical space enabled by synthetic organic chemistry. While chemical biologists and drug discovery scientists have yet to equal the exquisite selectivity of biochemical PTM, structure-guided design and ever improving computational tools for predicting chemical reactivity and ligand binding portend a bright future for cysteine-reactive small molecules in chemical biology and drug discovery.

Acknowledgments The authors thank the reviewers for insightful comments and suggestions, the National Science Foundation for a Predoctoral Fellowship (KKH) and Pfizer Inc. for postdoctoral support (DMT).

Biography Author Manuscript

Michelle R. Arkin


Hallenbeck et al.

Page 14

Author Manuscript

LIST OF ABBREVIATIONS


BTK

Bruton’s Tyrosine Kinase

DTNB

5,5’-dithiobis-(2-nitrobenzoic acid)

EGFR

Epidermal Growth Factor Receptor

ERK

Extracellular-Signal-Regulated Kinase

FBDD

Fragment Based Drug Discovery

FGFR

Fibroblast Growth Factor Receptor

GSH

Glutathione

HCV

Hepatitis C Virus

HDAC

Histone Deacetylase

IL-2

Interleukin-2

JAK

Janus Kinase

LCMS

Liquid Chromatography Mass Spectrometry

MOA

Mechanism of Action

NMR

Nuclear Magnetic Resonance

PD

Pharmacodynamics

PDGFR

Platelet-Derived Growth Factor Receptor

PDK1

Phosphoinositide-Dependent Kinase-1

PK

Pharmacokinetics

PTM

Post-Translational Modification

PTP

Protein Tyrosine Phosphatase

RSK

Ribosomal s6 Kinase

SAHA

Suberoylanilide Hydroxamic Acid

VEGFR

Vascular Endothelial Growth Factor Recepotor

Author Manuscript

REFERENCES 1. King JL, Jukes TH. Non-Darwinian evolution. Science (New York, N.Y.). 1969; 164(3881):788– 798. 2. Requejo R, Hurd TR, Costa NJ, Murphy MP. Cysteine residues exposed on protein surfaces are the dominant intramito-chondrial thiol and may protect against oxidative damage. Febs J. 2010; 277(6): 1465–1480. [PubMed: 20148960]


Hallenbeck et al.

Page 15

Author Manuscript Author Manuscript Author Manuscript Author Manuscript

3. Leonard SE, Reddie KG, Carroll KS. Mining the Thiol Proteome for Sulfenic Acid Modifications Reveals New Targets for Oxidation in Cells. ACS Chem. Biol. 2009; 4(9):783–799. [PubMed: 19645509] 4. Majmudar JD, Martin BR. Strategies for profiling native S-nitrosylation. Biopolymers. 2014; 101(2):173–179. [PubMed: 23828013] 5. Weerapana E, Wang C, Simon GM, Richter F, Khare S, Dillon MBD, Bachovchin DA, Mowen K, Baker D, Cravatt BF. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature. 2010; 468(7325):790–795. [PubMed: 21085121] 6. Johnson DS, Weerapana E, Cravatt BF. Strategies for discovering and derisking covalent, irreversible enzyme inhibitors. Fut. Med. Chem. 2010; 2(6):949–964. 7. Mah R, Thomas JR, Shafer CM. Drug discovery considerations in the development of covalent inhibitors. Bioorg. Med. Chem. Lett. 2014; 24(1):33–39. [PubMed: 24314671] 8. David-Cordonnier M-H, Laine W, Joubert A, Tardy C, Goossens J-F, Kouach M, Briand G, Thi Mai HD, Michel S, Tillequin F, Koch M, Leonce S, Pierre A, Bailly C. Covalent binding to glutathione of the DNA-alkylating antitumor agent, S23906-1. Euro. J. Biochem. 2003; 270(13):2848–2859. 9. Bauer RA. Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies. Drug Discov. Today. 2015; 20(9):1061–1073. [PubMed: 26002380] 10. Singh J, Petter RC, Baillie TA, Whitty A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 2011; 10(4):307–317. [PubMed: 21455239] 11. Moghaddam MF, Tang Y, O’Brien Z, Richardson SJ, Bacolod M, Chaturedi P, Apuy J, Kulkarni A. A Proposed Screening Paradigm for Discovery of Covalent Inhibitor Drugs. Drug Metab. Lett. 2014; 8(1):19–30. [PubMed: 24628405] 12. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, Kris MG, Varmus H. Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase Domain. PLoS. Med. 2005; 2(3):e73. [PubMed: 15737014] 13. Yun C-H, Mengwasser KE, Toms AV, Woo MS, Greulich H, Wong K-K, Meyerson M, Eck MJ. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl. Acad. Sci. U.S.A. 2008; 105(6):2070–2075. [PubMed: 18227510] 14. Walter AO, Sjin RTT, Haringsma HJ, Ohashi K, Sun J, Lee K, Dubrovskiy A, Labenski M, Zhu Z, Wang Z, Sheets M, Martin TS, Karp R, van Kalken D, Chaturvedi P, Niu D, Nacht M, Petter RC, Westlin W, Lin K, Jaw-Tsai S, Raponi M, Dyke TV, Etter J, Weaver Z, Pao W, Singh J, Simmons AD, Harding TC, Allen A. Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. Cancer Discov. 2013; 3(12):1404–1415. [PubMed: 24065731] 15. Katakami N, Atagi S, Goto K, Hida T, Horai T, Inoue A, Ichinose Y, Koboyashi K, Takeda K, Kiura K, Nishio K, Seki Y, Ebisawa R, Shahidi M, Yamamoto N. LUX-Lung 4: A Phase II Trial of Afatinib in Patients With Advanced Non–Small-Cell Lung Cancer Who Progressed During Prior Treatment With Erlotinib, Gefitinib, or Both. J. Clin. Oncol. 2013; 31(27):3335–3341. [PubMed: 23816963] 16. Sequist LV, Besse B, Lynch TJ, Miller VA, Wong KK, Gitlitz B, Eaton K, Zacharchuk C, Freyman A, Powell C, Ananthakrishnan R, Quinn S, Soria J-C. Neratinib, an Irreversible Pan-ErbB Receptor Tyrosine Kinase Inhibitor: Results of a Phase II Trial in Patients With Advanced Non– Small-Cell Lung Cancer. J. Clin. Oncol. 2010; 28(18):3076–3083. [PubMed: 20479403] 17. Zhou W, Ercan D, Chen L, Yun C-H, Li D, Capelletti M, Cortot AB, Chirieac L, Iacob RE, Padera R, Engen JR, Wong K-K, Eck MJ, Gray NS, Janne PA. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature. 2009; 462(7276):1070–1074. [PubMed: 20033049] 18. ClinicalTrials.gov Search: Osimertinib. https://www.clinicaltri-als.gov/ct2/results? term=Osimertinib 19. ClinicalTrials.gov Search: Rociletinib. https://www.clinicaltrials.gov/ct2/results?term=Rociletinib 20. Jänne PA, Yang JC-H, Kim D-W, Planchard D, Ohe Y, Ramalingam SS, Ahn M-J, Kim S-W, Su W-C, Horn L, Haggstrom D, Felip E, Kim J-H, Frewer P, Cantarini M, Brown KH, Dickinson PA, Ghiorghiu S, Ranson M. AZD9291 in EGFR Inhibitor–Resistant Non–Small-Cell Lung Cancer. N. Engl. J. Med. 2015; 372(18):1689–1699. [PubMed: 25923549]


Hallenbeck et al.

Page 16


21. Sequist LV, Soria J-C, Goldman JW, Wakelee HA, Gadgeel SM, Varga A, Papadimitrakopoulou V, Solomon BJ, Oxnard GR, Dziadziuszko R, Aisner DL, Doebele RC, Galasso C, Garon EB, Heist RS, Logan J, Neal JW, Mendenhall MA, Nichols S, Piotrowska Z, Wozniak AJ, Raponi M, Karlovich CA, Jaw-Tsai S, Isaacson J, Despain D, Matheny SL, Rolfe L, Allen AR, Camidge DR. Rociletinib in EGFR-Mutated Non–Small-Cell Lung Cancer. N. Engl. J. Med. 2015; 372(18): 1700–1709. [PubMed: 25923550] 22. Engel J, Richters A, Getlik M, Tomassi S, Keul M, Termathe M, Lategahn J, Becker C, MayerWrangowski S, Grütter C, Uhlenbrock N, Krüll J, Schaumann N, Eppmann S, Kibies P, Hoffgaard F, Heil J, Menninger S, Ortiz-Cuaran S, Heuckmann JM, Tinnefeld V, Zahedi RP, Sos ML, Schultz-Fademrecht C, Thomas RK, Kast SM, Rauh D. Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach. J. Med. Chem. 2015; 58(17):6844– 6863. [PubMed: 26275028] 23. Gayko U, Fung M, Clow F, Sun S, Faust E, Price S, James D, Doyle M, Bari S, Zhuang SH. Development of the Bruton's tyrosine kinase inhibitor ibrutinib for B cell malignancies. Ann. N. Y. Acad. Sci. 2015; 1358(1):82–94. [PubMed: 26348626] 24. Pan Z, Scheerens H, Li S-J, Schultz BE, Sprengeler PA, Burrill LC, Mendonca RV, Sweeney MD, Scott KCK, Grothaus PG, Jeffery DA, Spoerke JM, Honigberg LA, Young PR, Dalrymple SA, Palmer JT. Discovery of Selective Irreversible Inhibitors for Bruton’s Tyrosine Kinase. Chem Med Chem. 2007; 2(1):58–61. [PubMed: 17154430] 25. O'Shea JJ, Kontzias A, Yamaoka K, Tanaka Y, Laurence A. Janus kinase inhibitors in autoimmune diseases. Ann. Rheum. Dis. 2013; 72(suppl 2):ii111–ii115. [PubMed: 23532440] 26. Goedken ER, Argiriadi MA, Banach DL, Fiamengo BA, Foley SE, Frank KE, George JS, Harris CM, Hobson AD, Ihle DC, Marcotte D, Merta PJ, Michalak ME, Murdock SE, Tomlinson MJ, Voss JW. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol. J. Biol. Chem. 2015; 290(8):4573–4589. [PubMed: 25552479] 27. Tanaka H, Nishida K, Sugita K, Yoshioka T. Antitumor efficacy of hypothemycin, a new Rassignaling inhibitor. Jpn. J. Cancer. Res. 1999; 90(10):1139–1145. [PubMed: 10595743] 28. Barluenga S, Jogireddy R, Koripelly GK, Winssinger N. In Vivo Efficacy of Natural ProductInspired Irreversible Kinase Inhibitors. Chem Bio Chem. 2010; 11(12):1692–1699. 29. Winssinger N, Barluenga S. Chemistry and biology of resorcylic acid lactones. Chem. Commun. 2007; (1):22–36. 30. Schirmer A, Kennedy J, Murli S, Reid R, Santi DV. Targeted covalent inactivation of protein kinases by resorcylic acid lactone polyketides. Proc. Natl. Acad. Sci. U.S.A. 2006; 103(11):4234– 4239. [PubMed: 16537514] 31. ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/NCT02418000 32. Kumar V, Schuck EL, Pelletier RD, Farah N, Condon KB, Ye M, Rowbottom C, King BM, Zhang Z-Y, Saxton PL, Wong YN. Pharmacokinetic characterization of a natural product–inspired novel MEK1 inhibitor E6201 in preclinical species. Cancer Chemother. Pharmacol. 2011; 69(1):229– 237. [PubMed: 21698359] 33. Hagel M, Niu D, St Martin T, Sheets MP, Qiao L, Bernard H, Karp RM, Zhu Z, Labenski MT, Chaturvedi P, Nacht M, Westlin WF, Petter RC, Singh J. Selective irreversible inhibition of a protease by targeting a noncatalytic cysteine. Nat, Chem. Biol. 2011; 7(1):22–24. [PubMed: 21113170] 34. Daniel KB, Sullivan ED, Chen Y, Chan JC, Jennings PA, Fierke CA, Cohen SM. Dual-Mode HDAC Prodrug for Covalent Modification and Subsequent Inhibitor Release. J. Med. Chem. 2015; 58(11):4812–4821. [PubMed: 25974739] 35. Visscher M, Arkin MR, Dansen TB. Covalent targeting of acquired cysteines in cancer. Curr. Opin. Chem. Biol. 2016; 30:61–67. [PubMed: 26629855] 36. Kozakov D, Grove LE, Hall DR, Bohnuud T, Mottarella SE, Luo L, Xia B, Beglov D, Vajda S. The FTMap family of web servers for determining and characterizing ligand-binding hot spots of proteins. Nat. Protocols. 2015; 10(5):733–755. [PubMed: 25855957] 37. Beuming T, Che Y, Abel R, Kim B, Shanmugasundaram V, Sherman W. Thermodynamic analysis of water molecules at the surface of proteins and applications to binding site prediction and characterization. Proteins: Struct., Funct., Bioinf. 2012; 80(3):871–883.


Hallenbeck et al.

Page 17


38. Arkin MR, Randal M, DeLano WL, Hyde J, Luong TN, Oslob JD, Raphael DR, Taylor L, Wang J, McDowell RS, Wells JA, Braisted AC. Binding of small molecules to an adaptive protein–protein interface. Proc. Natl. Acad. Sci. U.S.A. 2003; 100(4):1603–1608. [PubMed: 12582206] 39. Dong GQ, Calhoun S, Fan H, Kalyanaraman C, Branch MC, Mashiyama ST, London N, Jacobson MP, Babbitt PC, Shoichet BK, Armstrong RN, Sali A. Prediction of Substrates for Glutathione Transferases by Covalent Docking. J. Chem. Inform. Modeling. 2014; 54(6):1687–1699. 40. London N, Miller RM, Krishnan S, Uchida K, Irwin JJ, Eidam O, Gibold L, Cimermančič P, Bonnet R, Shoichet BK, Taunton J. Covalent docking of large libraries for the discovery of chemical probes. Nat. Chem. Biol. 2014; 10(12):1066–1072. [PubMed: 25344815] 41. Kathman SG, Xu Z, Statsyuk AV. A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases. J. Med. Chem. 2014; 57(11):4969–4974. [PubMed: 24870364] 42. Miller RM, Paavilainen VO, Krishnan S, Serafimova IM, Taunton J. Electrophilic Fragment-Based Design of Reversible Covalent Kinase Inhibitors. J. Am. Chem. Soc. 2013; 135(14):5298–5301. [PubMed: 23540679] 43. Hopkins, AL., Groom, CR., Alex, A. Drug Discov. Today. Vol. 9. England: 2004. Ligand efficiency: a useful metric for lead selection; p. 430-431. 44. Murray CW, Rees DC. The rise of fragment-based drug discovery. Nat. Chem. 2009; 1(3):187– 192. [PubMed: 21378847] 45. Kumar A, Zhang AVAKYJ. Fragment Based Drug Design: From Experimental to Computational Approaches. Curr. Med. Chem. 2012; 19(30):5128–5147. [PubMed: 22934764] 46. Joseph-McCarthy D, Campbell AJ, Kern G, Moustakas D. Fragment-Based Lead Discovery and Design. J. Chem. Inform. Modeling. 2014; 54(3):693–704. 47. Doak BC, Morton CJ, Simpson JS, Scanlon MJ. Design and Evaluation of the Performance of an NMR Screening Fragment Library. Aust. J. Chem. 2013; 66(12):1465–1472. 48. Francis CL, Kenny PW, Dolezal O, Saubern S, Kruger M, Savage GP, Peat TS, Ryan JH. Construction of the CSIRO Fragment Library. Aust. J. Chem. 2013; 66(12):1473–1482. 49. Rahman, A. Frontiers in Drug Design and Discovery. Bentham Science Publishers; 2007. 50. Jhoti H, Williams G, Rees DC, Murray CW. The 'rule of three' for fragment-based drug discovery: where are we now? Nat. Rev. Drug Discov. 2013; 12(8):644–644. [PubMed: 23845999] 51. Congreve M, Carr R, Murray C, Jhoti H. A ‘Rule of Three’ for fragment-based lead discovery? Drug Discov. Today. 2003; 8(19):876–877. 52. Hajduk PJ, Greer J. A decade of fragment-based drug design: strategic advances and lessons learned. Nat. Rev. Drug Discov. 2007; 6(3):211–219. [PubMed: 17290284] 53. Hung AW, Ramek A, Wang Y, Kaya T, Wilson JA, Clemons PA, Young DW. Route to threedimensional fragments using diversity-oriented synthesis. Proc. Natl. Acad. Sci. U.S.A. 2011; 108(17):6799–6804. [PubMed: 21482811] 54. Dahlin JL, Nissink JWM, Strasser JM, Francis S, Higgins L, Zhou H, Zhang Z, Walters MA. PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed during a Sulfhydryl-Scavenging HTS. J. Med. Chem. 2015; 58(5):2091–2113. [PubMed: 25634295] 55. Baell J, Walters MA. Chemistry: Chemical con artists foil drug discovery. Nature. 2014; 513(7519):481–483. [PubMed: 25254460] 56. McGovern SL, Caselli E, Grigorieff N, Shoichet BK. A Common Mechanism Underlying Promiscuous Inhibitors from Virtual and High-Throughput Screening. J. Med. Chem. 2002; 45(8): 1712–1722. [PubMed: 11931626] 57. Oprea TI, Davis AM, Teague SJ, Leeson PD. Is There a Difference between Leads and Drugs? A Historical Perspective. J. Chem. Inform. Comput. Sci. 2001; 41(5):1308–1315. 58. Baell JB, Holloway GA. New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays. J. Med. Chem. 2010; 53(7):2719–2740. [PubMed: 20131845] 59. Kwarcinski FE, Steffey ME, Fox CC, Soellner MB. Discovery of Bivalent Kinase Inhibitors via Enzyme-Templated Fragment Elaboration. ACS. Med. Chem. Lett. 2015; 6(8):898–901. [PubMed: 26286460]


Hallenbeck et al.

Page 18


60. Allen CE, Curran PR, Brearley AS, Boissel V, Sviridenko L, Press NJ, Stonehouse JP, Armstrong A. Efficient and facile synthesis of acrylamide libraries for protein-guided tethering. Org. Lett. 2015; 17(3):458–460. [PubMed: 25582165] 61. Flanagan ME, Abramite JA, Anderson DP, Aulabaugh A, Dahal UP, Gilbert AM, Li C, Montgomery J, Oppenheimer SR, Ryder T, Schuff BP, Uccello DP, Walker GS, Wu Y, Brown MF, Chen JM, Hayward MM, Noe MC, Obach RS, Philippe L, Shanmugasundaram V, Shapiro MJ, Starr J, Stroh J, Che Y. Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. J. Med. Chem. 2014; 57(23): 10072–10079. [PubMed: 25375838] 62. Schwöbel JAH, Koleva YK, Enoch SJ, Bajot F, Hewitt M, Madden JC, Roberts DW, Schultz TW, Cronin MT. D. Measurement and Estimation of Electrophilic Reactivity for Predictive Toxicology. Chem. Rev. 2011; 111(4):2562–2596. [PubMed: 21401043] 63. Enoch SJ, Ellison CM, Schultz TW, Cronin MT. D. A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity. Crit. Rev. Toxicol. 2011; 41(9): 783–802. [PubMed: 21809939] 64. Nishino M, Choy JW, Gushwa NN, Oses-Prieto JA, Koupparis K, Burlingame AL, Renslo AR, McKerrow JH, Taunton J. Hypothemicin, a fungal natural product, identifies therapeutic targets in Trypanosoma brucei. eLife. 2013; 2:e00712. [PubMed: 23853713] 65. Choy JW, Bryant C, Calvet CM, Doyle PS, Gunatilleke SS, Leung SSF, Ang KKH, Chen S, Gut J, Oses-Prieto JA, Johnston JB, Arkin MR, Burlingame AL, Taunton J, Jacobson MP, McKerrow JM, Podust LM, Renslo AR. Chemical–biological characterization of a cruzain inhibitor reveals a second target and a mammalian off-target. Beilstein. J. Org. Chem. 2013; 9:15–25. [PubMed: 23400640] 66. Erlanson DA, Braisted AC, Raphael DR, Randal M, Stroud RM, Gordon EM, Wells JA. Sitedirected ligand discovery. Proc. Natl. Acad. Sci. U.S.A. 2000; 97(17):9367–9372. [PubMed: 10944209] 67. Lodge JM, Justin Rettenmaier T, Wells JA, Pomerantz WC, Mapp AK. FP tethering: a screening technique to rapidly identify compounds that disrupt protein-protein interactions. MedChemComm. 2014; 5(3):370–375. 68. Erlanson DA, Lam JW, Wiesmann C, Luong TN, Simmons RL, DeLano WL, Choong IC, Burdett MT, Flanagan WM, Lee D, Gordon EM, O'Brien T. In situ assembly of enzyme inhibitors using extended tethering. Nat. Biotech. 2003; 21(3):308–314. 69. Ostrem JM, Peters U, Sos ML, Wells JA, Shokat KM. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature. 2013; 503(7477):548–551. [PubMed: 24256730] 70. Erlanson DA, Arndt JW, Cancilla MT, Cao K, Elling RA, English N, Friedman J, Hansen SK, Hession C, Joseph I, Kumaravel G, Lee W-C, Lind KE, McDowell RS, Miatkowski K, Nguyen C, Nguyen TB, Park S, Pathan N, Penny DM, Romanowski MJ, Scott D, Silvian L, Simmons RL, Tangonan BT, Yang W, Sun L. Discovery of a potent and highly selective PDK1 inhibitor via fragment-based drug discovery. Bioorg. Med. Chem. Lett. 2011; 21(10):3078–3083. [PubMed: 21459573] 71. Raimundo BC, Oslob JD, Braisted AC, Hyde J, McDowell RS, Randal M, Waal ND, Wilkinson J, Yu CH, Arkin MR. Integrating Fragment Assembly and Biophysical Methods in the Chemical Advancement of Small-Molecule Antagonists of IL-2: An Approach for Inhibiting Protein–Protein Interactions†. J. Med. Chem. 2004; 47(12):3111–3130. [PubMed: 15163192] 72. Adjei AA. Blocking Oncogenic Ras Signaling for Cancer Therapy. J. Natl. Cancer Inst. 2001; 93(14):1062–1074. [PubMed: 11459867] 73. Wang Y, Kaiser CE, Frett B, Li H-Y. Targeting Mutant KRAS for Anticancer Therapeutics: A Review of Novel Small Molecule Modulators. J. Med. Chem. 2013; 56(13):5219–5230. [PubMed: 23566315] 74. Patricelli MP, Janes MR, Li L-S, Hansen R, Peters U, Kessler LV, Chen Y, Kucharski JM, Feng J, Ely T, Chen JH, Firdaus SJ, Babbar A, Ren P, Liu Y. Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State. Cancer Discov. 2016 75. Hunter JC, Gurbani D, Ficarro SB, Carrasco MA, Lim SM, Choi HG, Xie T, Marto JA, Chen Z, Gray NS, Westover KD. In situ selectivity profiling and crystal structure of SML-8-73-1, an active Curr Top Med Chem. Author manuscript; available in PMC 2017 February 22.

Hallenbeck et al.

Page 19

Author Manuscript Author Manuscript Author Manuscript

site inhibitor of oncogenic K-Ras G12C. Proc. Natl. Acad. Sci. U.S.A. 2014; 111(24):8895–8900. [PubMed: 24889603] 76. Lim SM, Westover KD, Ficarro SB, Harrison RA, Choi HG, Pacold ME, Carrasco M, Hunter J, Kim ND, Xie T, Sim T, Jänne PA, Meyerson M, Marto JA, Engen JR, Gray NS. Therapeutic Targeting of Oncogenic K-Ras by a Covalent Catalytic Site Inhibitor. Angewandte Chemie Intl. Edition. 2014; 53(1):199–204. 77. Copeland RA, Pompliano DL, Meek TD. Drug-target residence time and its implications for lead optimization. Nat. Rev. Drug Discov. 2006; 5(9):730–739. [PubMed: 16888652] 78. Serafimova IM, Pufall MA, Krishnan S, Duda K, Cohen MS, Maglathlin RL, McFarland JM, Miller RM, Frodin M, Taunton J. Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles. Nat. Chem. Biol. 2012; 8(5):471–476. [PubMed: 22466421] 79. Bradshaw JM, McFarland JM, Paavilainen VO, Bisconte A, Tam D, Phan VT, Romanov S, Finkle D, Shu J, Patel V, Ton T, Li X, Loughhead DG, Nunn PA, Karr DE, Gerritsen ME, Funk JO, Owens TD, Verner E, Brameld KA, Hill RJ, Goldstein DM, Taunton J. Prolonged and tunable residence time using reversible covalent kinase inhibitors. Nat. Chem. Biol. 2015; 11(7):525–531. [PubMed: 26006010] 80. Hardy JA, Lam J, Nguyen JT, O'Brien T, Wells JA. Discovery of an allosteric site in the caspases. Proc. Natl. Acad. Sci. U.S.A. 2004; 101(34):12461–12466. [PubMed: 15314233] 81. Scheer JM, Romanowski MJ, Wells JA. A common allosteric site and mechanism in caspases. Proc. Natl. Acad. Sci. U.S.A. 2006; 103(20):7595–7600. [PubMed: 16682620] 82. Datta D, Scheer JM, Romanowski MJ, Wells JA. An Allosteric Circuit in Caspase-1. J. Mol. Biol. 2008; 381(5):1157–1167. [PubMed: 18590738] 83. Hardy JA, Wells JA. Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes. J. Biol. Chem. 2009; 284(38):26063–26069. [PubMed: 19581639] 84. Gao J, Sidhu SS, Wells JA. Two-state selection of conformation- specific antibodies. Proc. Natl. Acad. Sci. U.S.A. 2009; 106(9):3071–3076. [PubMed: 19208804] 85. DeLano WL, Ultsch MH, de AM, Vos, Wells JA. Convergent Solutions to Binding at a ProteinProtein Interface. Science (New York, N.Y.). 2000; 287(5456):1279–1283. 86. Wells JA, McClendon CL. Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature. 2007; 450(7172):1001–1009. [PubMed: 18075579] 87. Gold MG, Barford D, Komander D. Lining the pockets of kinases and phosphatases. Curr. Opin. Structural Biol. 2006; 16(6):693–701. 88. Sadowsky JD, Burlingame MA, Wolan DW, McClendon CL, Jacobson MP, Wells JA. Turning a protein kinase on or off from a single allosteric site via disulfide trapping. Proc. Natl. Acad. Sci. U.S.A. 2011; 108(15):6056–6061. [PubMed: 21430264] 89. Chio CM, Lim CS, Bishop AC. Targeting a Cryptic Allosteric Site for Selective Inhibition of the Oncogenic Protein Tyrosine Phosphatase Shp2. Biochemistry. 2015; 54(2):497–504. [PubMed: 25519989] 90. Chio CM, Yu X, Bishop AC. Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases. Bioorg. Med. Chem. 2015; 23(12):2828–2838. [PubMed: 25828055] 91. Bowman GR, Bolin ER, Hart KM, Maguire BC, Marqusee S. Discovery of multiple hidden allosteric sites by combining Markov state models and experiments. Proc. Natl. Acad. Sci. U.S.A. 2015; 112(9):2734–2739. [PubMed: 25730859]

Author Manuscript Curr Top Med Chem. Author manuscript; available in PMC 2017 February 22.

Hallenbeck et al.

Page 20

Author Manuscript Fig. (1).

Author Manuscript

Schematic of Covalent Enzyme Inhibition. An initial non-covalent binding event brings the cysteine sulfhydryl group in proximity to the warhead X, driving covalent bond formation. The covalent bond can be irreversible (e.g vinyl sulfonamide), where k−2 is zero, reversible (e.g. another sulfhydryl group) where k−2 depends on reaction conditions or slowly reversible (e.g. cyanoacrylamide), where k−2 depends on warhead reactivity.

Author Manuscript Author Manuscript Curr Top Med Chem. Author manuscript; available in PMC 2017 February 22.

Hallenbeck et al.

Page 21

Author Manuscript Author Manuscript Author Manuscript Fig. (2).

Author Manuscript

Deriving Covalent Inhibitors from Known Scaffolds. A–B) Non-covalent 1st generation EGFR inhibitor Gefitinib (purple) overlaid with covalent 2nd generation inhibitor Afatinib (white). T790, the eventual site of resistance mutation, is shown. B–C) Natural product Hypothemycin bound to ERK2 and synthetic drug candidate E6201. E–F) Telaprevir (purple) overlaid with covalent derivative Compound 3 (white), which binds to conserved Cys159.


Hallenbeck et al.

Page 22

Author Manuscript Author Manuscript Author Manuscript

Fig. (3).

Applications of Disulfide Tethering. A–C) K-Ras G12C mutation targeted by an optimized tethering hit (white; B), validating a new pocket; chemical optimization of electrophilic analogs led to an irreversible inhibitor (purple; C) with nM potency. D,E) Caspase-1 zymogen dimer (monomers colored tan/purple) bound to two DICA molecules at Cys290. F,G) Caspase-7 dimer (monomer surface colored tan/purple) bound to two interacting copies of Compound 34 (F). H–J) PDK1 bound to activator JS30 (purple; I) and inhibitor 1F8 (white; J) at the PIF-pocket. The activator shifts the regulatory C-helix down toward the active site where an active-site inhibitor BIM-II is bound (purple).

Author Manuscript Curr Top Med Chem. Author manuscript; available in PMC 2017 February 22.

Hallenbeck et al.

Page 23

Author Manuscript Author Manuscript Fig. (4).

Slowly Reversible Cyanoacrylamide Inhibitors of BTK. The core Ibrutinib scaffold was systematically substituted with three cyanoacrylamide warheads: 1 Me, 2 i-Pr, and 3 t-Bu. Compounds 1–3 had similar EC50 values, but vastly differing residence times (τ) that correlated with increasing steric demand (t-Bu > i-Pr > Me).

Author Manuscript Author Manuscript Curr Top Med Chem. Author manuscript; available in PMC 2017 February 22.

Targeting epigenetics in drug discovery.

Drug Discovery Targeting Bromodomain-Containing Protein 4.

Rectifying cancer drug discovery through network pharmacology.

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

Targeting iron metabolism in drug discovery and delivery.

Approaches of targeting Rho GTPases in cancer drug discovery.

Targeting dengue virus NS4B protein for drug discovery.

Targeting GABAB receptors for anti-abuse drug discovery.

Overcoming melanoma drug resistance through metabolic targeting?

Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis.

Identifying Functional Cysteine Residues in the Mitochondria.

Insights into drug discovery from natural products through structural modification.

Driving Structure-Based Drug Discovery through Cosolvent Molecular Dynamics.

Drug discovery through stem cell-based organoid models.

Discovery of BI 224436, a Noncatalytic Site Integrase Inhibitor (NCINI) of HIV-1.

Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues.

Drug discovery FAQs: workflows for answering multidomain drug discovery questions.

Chemical Protein Ubiquitylation with Preservation of the Native Cysteine Residues.

On methylene-bridged cysteine and lysine residues in proteins.

Specific enzymic cleavage of polypeptides at cysteine residues.

Cysteine residues in the active site of Corynebacterium sarcosine oxidase.

Lysosomal handling of cystine residues: stoichiometry of cysteine involvement.

Protein isoprenylation and methylation at carboxyl-terminal cysteine residues.

A hybrid platinum drug dichloroacetate-platinum(II) overcomes cisplatin drug resistance through dual organelle targeting.